Most reforestation is now done using seedlings grown in nurseries, if the Douglas-fir region of the Pacific Northwest or southern pine region of the Southeastern United States may be taken as examples. Most seed for the nurseries is provided by seed orchards that, in some cases, are now a third generation of selected trees. Most of this orchard produced seed has been open pollinated and much of the pollen comes from wild trees located outside the orchard. For this reason, the maximum genetic gain has not been reached. Some full sib seed is produced, in which both cone and pollen parents are known. However, this is in far too short supply and is much too expensive for any but very specialized applications. Some is effectively bulked up as nursery stock used for the production of rooted cuttings. While still in the early stages of commercial production of reforestation stock, selected seed may also be bulked up by tissue culture methods.
The optimum time to harvest cones for their contained seeds has always been a problem in forest tree nurseries. Too early harvest will yield immature seed that may have poor germination or result in trees having less than the desired vigor. Too late harvest may result in significant seed loss from cones opening. In analogous fashion, it is always problematical when somatic embryos have obtained the maturity that will lead to good germination.
In seeds naturally grown on plants, "maturity" corresponds to the time when the seed dries and dehisces from the mother plant. This point in time may be assumed to represent the seed (zygotic) embryo biochemical maturity as well as morphological maturity. Morphological maturity may be defined as the time when cell division in the embryo ceases. Biochemical maturity is seen as the time when accumulation of storage products is complete and the seed is ready to enter dormancy. Essentially, the genetically pre-programmed development and environmental responses of the embryo and mother plant dictate maturity. It is only necessary to harvest the mature seed at the proper time and treat it optimally after harvest. However, this is not the case with somatic embryos. Scientists must dictate the timing and protocol of every shift in hormones, media composition, water potential, photoperiod, and temperature. In somatic embryos, there is no period of quiescence which clearly demarcates maturation from readiness for germination in natural seed.
Tissue culture of somatic embryos of coniferous species has received heavy emphasis in recent years as a means of supplying genetically superior reforestation stock. A seed, or the contained embryo, is placed on a culture medium with appropriate mineral, vitamin and hormone nutrients. When initiation is successful, a gelatinous mass will form containing a multiplicity of immature embryos genetically identical to the parent embryo. This mass is then transferred to another medium, often reduced in hormone content, for maintenance and multiplication of the embryos. The culture, or a portion of it, is again transferred to a growth or development medium where the embryos mature to a point where they morphologically resemble natural zygotic embryos. These embryos may then be placed on a germination medium where the resulting plantlets are allowed to grow until they can be transferred to soil. Alternatively the embryos can be placed in manufactured seeds and be planted directly in soil. U.S. Pat. No. 5.036,007 to Gupta et al. is exemplary of the tissue culture methods used for coniferous species.
Unfortunately, tissue culture is known to be a highly unpredictable science. It is well accepted that there is a high level of unpredictability between and even within genera in morphological and biochemical development. This is particularly true comparing the angiosperms and gymnosperms. It has been shown many times that what might be true for alfalfa or soy beans is not necessarily true at all for pine or Douglas-fir trees. This applies even below the species level. A culturing protocol that works well for one genotype may not be optimum for another genotype obtained from the same cone.
Appearance has heretofore been used as a criterion of maturity for somatic embryos produced by tissue culture; i.e., if a somatic embryo looked like a mature zygotic embryo it was presumed to be mature. Unfortunately, this crude tool has proved to be highly undependable. The level of "storage products" has also received some attention as a maturity indicator. Storage products are generally defined as the protein, lipid, and carbohydrate materials needed to support the embryo during and immediately after germination. One group of workers has found that inducing an exaggerated level of triacylglycerides in conifer somatic embryos improves germination (Attree et al., U.S. Pat. No. 5,464,769).
For many angiosperm species at least, it is now known that embryo biochemical maturity lags behind morphological maturity by a significant period of time. It is during this time interval, between morphological and biochemical maturity, that oligosaccharides of the raffinose series develop. As examples, Gorecki et al. (1997) and Obendorf (1997) discuss the importance of the oligosaccharides and note that certain cyclitols and galactosyl cyclitols appear to be formed in the time period between morphological and biochemical maturity. The presence of a protein group called dehydrins, generally formed after accumulation of storage products is complete, is also an indicator of angiosperm readiness to germinate. Published application WO 98/48279, commonly assigned with the present one, describes a method of estimating maturity of conifer somatic embryos by analysis of their oligosaccharides. This application is herein incorporated in its entirety by reference.
Conifer seed, whether full sib seed from nurseries or seed gathered in the wild, will have all of the genetic diversity of the parent trees. Some seeds will produce trees with more desirable characteristics than other seeds taken from the same cone. Tissue culture offers a way to select the best trees before major plantings have been made in the forest. Embryos at the maintenance phase may be frozen in liquid nitrogen and stored while a small sample of the clone is further cultured and outplanted. After three to five years the characteristics of the clone are readily apparent and the decision can then be made whether or not to remove the remainder of the embryos from cryogenic storage for larger scale propagation.
The requirements of an embryo during maturation are completely different and virtually opposite to the requirements of an embryo during germination. In early and mid development, morphology is the outcome or result of changes that have taken place at the biochemical level. However, it does not reveal all of them, particularly at the critical juncture between biochemical maturity and readiness to germinate. More precise biochemical tools to signal these changes would be extremely helpful to seed orchardists and to the scientists working with somatic embryogenesis. In the case of the latter group, the availability of such tools would be of great value, both for determining readiness for germination and for developing culture medium protocols to optimally achieve such readiness. It would allow them to identify and develop needed protocol changes and the timing of their imposition.
The literature on the biochemistry of the late development period in small seed crops has noted the presence of sugar alcohols and presumed that they serve in the acquisition of desiccation tolerance. In the discussion that follows, literature citations list only the lead author and date of publication. Full citations are given in the attached bibliography at the end of the specification.
Horbowicz et al. (1998) characterized a major soluble carbohydrate in buckwheat as O-.alpha.-D-galactopyranosyl-(1.fwdarw.2)-D-chiro-inositol or fagopyritol B1. They suggest that accumulation of fagopyritol was associated with acquisition of desiccation tolerance during the latter part of seed development. D-chiro-inositol and myo-inositol were present throughout seed development but pinitol or other O-methylated inositols were not found at any time.
Obendorf et al. (1998) note that fagopyritol and galactopinitol accumulate in soybean axis tissues in parallel with stachyose accumulation, in association with the advent of desiccation tolerance. Phillipy (1998) notes various inositol derivatives in immature soybean tissue but does not discuss these in the context of embryo maturation. Peterbauer et al. (1998) note the synthesis of a galactosylcyclitol by stachyose synthase extracted from adzuki bean. Additionally, the enzyme catalyzed the galactinol-dependent synthesis of galactosylononitol from D-ononitol. Murphy et al. (1998), in an analysis of the soluble sugars and hydrolytic enzymes in sugar pine embryos before and during germination, made no note of any sugar alcohols other than pinitol, which was present at a constant low level throughout their seed incubation period. Kuo et al. (1997) identified pinitol as a major cyclitol in developing soybean. Pinitol was seen to decrease sharply as raffinose polysaccharides accumulated. Gorecki et al. (1997) investigated cyclitols in lupine seeds during their period of maturation. They found D-pinitol, D-chiro-inositol, and myo-inositol, to be predominant in the early stages of seed growth. During maturation raffinose family oligosaccharides accumulated as did the galactosyl cyclitols; e.g., fagopyritols and galactopinitols. The increase of these latter compounds appeared to correlate with seed germinability after desiccation.
Chien et al. (1996) identified a galactopinitol in the late development stages of the seeds of leucaena seeds. Leucaena is a small leguminous tree and appears to be the only tree seed which has been investigated for cyclitols. Interestingly, the galactopinitol they found decreased during maturation while stachyose concentration increased. Horbowicz et al. (1995) looked at the maturation of alfalfa seeds and somatic embryos. They note that during maturation and desiccation of somatic embryos, changes in soluble carbohydrates are similar to those of seeds, with the notable exception of the lack of pinitol and galactosyl pinitols in the somatic embryos. Horbowicz et al. (1994) studied carbohydrates in the axes, cotyledons, embryos, and seeds of 19 species in 7 families in regard to desiccation tolerance. Considerable differences were found. Legumes tended to accumulate stachyose series oligosaccharides whereas many other species accumulated galactosyl derivatives of cyclitols. One species (castor bean) accumulated galactinol whereas buckwheat accumulated galacto-chiro-inositol (fagopyritol). The authors proposed that galactinol and galacto-chiro-inositol functioned in the same manner as raffinose in giving desiccation tolerance. The paper is a handy reference to the chemical structures of the various cyclitols.
Bernabe et al. (1993) detail a method used to identify the structure of ciceritol, a pseudotrisaccharide found in lentils. Kuo (1992) noted that galactinol (1-O-.alpha.-D-galactopyranosyl-D-myo-inositol) is thought to be an essential intermediate in the biosynthesis of the raffinose series polysaccharides in plant tissues. This paper describes a method for extracting reference quantities of galactinol from castor oilseed meal. Baron (1970) studied the germination of sugar pine seeds. This early paper noted that during stratification, fat content decreased as did raffinose and stachyose. These oligosaccharides completely disappeared during germination. The only cyclitol examined was myo-inositol which was found to increase as germination progressed.
Thus, it is seen that the literature regarding the biochemistry of the late development period has been essentially silent as to the presence and presumed function of the sugar alcohols in conifer seeds and/or embryos.